Fibrosis and wound healing 2013
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Fibrosis and wound healing 2013

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Fibrosis and wound healing 2013 Fibrosis and wound healing 2013 Presentation Transcript

  • Sample & Assay Technologies Any Questions ??? Ask now or contact support support@SABiosciences.com 1-888-503-3187 International customers: SABio@Qiagen.com Webinar related questions: Qiawebinars@qiagen.com Uncovering the mechanisms of wound healing and fibrosis
  • Sample & Assay Technologies Legal disclaimers RT2 Profiler PCR Arrays and Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, please see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.QIAGEN.com, or can be requested from QIAGEN Technical Services or your local distributor. Title, Location, Date 2
  • Sample & Assay Technologies Background: process of wound healing Wound healing — a 4-step process Step 1: Hemostasis (clotting) Step 2: Inflammation Vasoconstriction Enhanced blood vessel permeability due to release of histamine and other factors Coagulation cascade is initiated by interaction of coagulation factor FVII with TF Von Willebrand factor helps platelets bind at wound sites, where they activate and degranulate Step 3: Proliferative phase Inflammatory cells infiltrate (PMN, macrophages) and kill any microbes accompanying the injury Leukocytes produce cytokines, chemokines, and growth factors, some of which (IL-1beta, TGF-beta, TNF) recruit fibroblasts Step 4: Wound closing/tissue remodeling Fibroblasts are recruited and activated, and secrete Wound contraction via myofibroblasts at the edges ECM components (type III collagen, fibronectin) Formation of granulation tissue (fibroblasts, inflammatory cells, new blood vessels, fibronectin, hyaluronan, collagen, endothelial cells) Type III collagen is replaced by Type I, and fibers are rearranged and crosslinked. Remodeling will continue for weeks to months. Epithelialization Title, Location, Date 3
  • Sample & Assay Technologies Backgrounds: wound healing and fibrosis Uncontrolled wound healing response results in fibrosis Fibrosis develops if any stage in the tissue repair program is dysregulated. Chronic inflammation The tissue-damaging agent is not removed The repair process is not regulated properly Review, Integrating mechanisms of pulmonary fibrosis. JEM Vol. 208, No. 7 Title, Location, Date 4
  • Sample & Assay Technologies Background: Key components of wound healing/fibrosis Cells/cell fragments: Epithelial and endothelial cells Platelets Fibroblasts / Myofibroblasts Inflammatory cells (macrophages, neutrophils) Proteins: ECM components (Collagens, fibronectin, etc.) Proteases (MMPs, collagenase) Growth factors (TGF-beta, PDGF) Inflammatory mediators (histamine) Cytokines (TNF-alpha, IL-1-beta) Intracellular signaling pathways (NFkappaB, JAK/STAT, more) Title, Location, Date 5
  • Sample & Assay Technologies Background: Fibrosis and wound healing Fibrosis affects a wide range of tissues, including: Lungs (idiopathic pulmonary fibrosis, cystic fibrosis) Heart (post-myocardial infarction, endomyocardial fibrosis) Liver (cirrhosis) Skin (Scleroderma, nephrogenic systemic fibrosis) Joints (arthrofibrosis) Kidney (renal fibrosis) Title, Location, Date 6
  • Sample & Assay Technologies Background: Fibrosis and diseases Cardiac fibrosis Can occur in response to left ventricular pressure-overload, as “reactive interstitial fibrosis”, which leads to cardiac hypertrophy and necrosis Alternatively, “replacement fibrosis” could occur in response to myocardial infarction, inflammation, and myocyte death. Systemic scleroderma - skin Autoimmune disease of the skin and, in some cases, the internal organs Arteriole endothelial and smooth muscle cell apoptosis, followed by inflammation and fibrosis Cause is unknown, but skin fibrosis can be treated Liver cirrhosis Develops as a result of chronic liver disease (alcoholic, fatty liver disease, autoimmune disease, or hepatitis virus, etc.), or idiopathic Title, Location, Date 7
  • Sample & Assay Technologies Background: Fibrosis and diseases Pulmonary fibrosis Idiopathic (possibly linked to Surfactant protein C mutation) or as a result of injury or disease, including: Inhalation of particulates and gases Smoking Infections Drugs (bleomycin, amiodarone, etc.) Involvement by inflammatory mediators such as TNF, IL-beta, and IL-17, has been implicated, as well as Th2 cytokines such as IL-13 and IL-4 Review, Integrating mechanisms of pulmonary fibrosis. JEM Vol. 208, No. 7 Title, Location, Date 8
  • Sample & Assay Technologies Pulmonary fibrosis: role of T helper cells and macrophages Review, Integrating mechanisms of pulmonary fibrosis. JEM Vol. 208, No. 7 Title, Location, Date 9
  • Sample & Assay Technologies Researching the causes of fibrosis Three recent case studies in scleroderma and cardiac fibrosis Scleroderma and cytokines SOCS3 and myocardial infarction Matrix metalloproteinases and LV remodeling Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts. Nakashima, T. et al. (2012) Journal of Immunology Cardiac-specific deletion of SOCS-3 prevents development of left ventricular remodeling after acute myocardial infarction. Oba, T. et al. (2012) Journal of the American College of Cardiology Pressure overloaddependent membrane type Imatrix metalloproteinase induction: relationship to LV remodeling and fibrosis. Zile, M.R. et al. (2012) American Journal of Physiology, Heart and Circulation Physiology Used RT2 Profiler PCR Array for Human Extracellular Matrix and Adhesion Molecules Used RT2 Profiler PCR Array for Mouse Common Cytokines Used a Custom RT2 Profiler PCR Array. Title, Location, Date 10
  • Sample & Assay Technologies RT2 Profiler PCR Arrays Pathway-focused gene expression profiling - 84 (or 370) real-time PCR assays for genes related to specific pathways Gene lists chosen by our experts – bioinformatics and text mining Each assay is wet-lab tested for specificity and sensitivity More than 140 pathways available, including many for fibrosis-related processes Integrated controls for normalization, reverse transcription, genomic DNA contamination, and the PCR process, plus free data analysis tools PCR Array Overview, Nov 15, 9:30am https://www2.gotomeeting.com/register/263258378 Title, Location, Date 11
  • Sample & Assay Technologies RT2 Profiler PCR Arrays for fibrosis and wound healing Fibrosis (for Human, Rat, Mouse, Rabbit, and more) http://sabiosciences.com/rt_pcr_product/HTML/PAHS-120Z.html Title, Location, Date 12
  • Sample & Assay Technologies RT2 Profiler PCR Arrays for fibrosis and wound healing Wound healing (for Human, Mouse, Rat, Pig, Rabbit, and more) http://sabiosciences.com/rt_pcr_product/HTML/PAHS-121Z.html Title, Location, Date 13
  • Sample & Assay Technologies RT2 Profiler PCR Arrays for fibrosis and wound healing ECM & Adhesion Molecules (for Human, Mouse, Rat) http://sabiosciences.com/rt_pcr_product/HTML/PAHS-013Z.html Title, Location, Date 14
  • Sample & Assay Technologies RT2 Profiler PCR Arrays for fibrosis and wound healing Additional pathways available Common Cytokines Inflammatory Cytokines & Receptors TGF-beta Signaling Pathway TGF-beta Signaling Targets Endothelial Cell Biology Epithelial-to-Mesenchymal Transition For complete list, see http://www.sabiosciences.com/ArrayList.php Other species and custom array Title, Location, Date 15
  • Sample & Assay Technologies An application example using RT2 Profiler PCR Arrays Cytokine expression changes in human PBMC on PMA/ionomycin treatment. Plot and Chart Format: Heat Map Scatter Plot Volcano Plot Clustergram Multigroup Plot Title, Location, Date 16
  • Sample & Assay Technologies Researching the causes of fibrosis 3 recent applications in scleroderma, cardiac and lung fibrosis Scleroderma and cytokines SOCS3 and myocardial infarction Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts. Nakashima, T. et al. (2012) Journal of Immunology Pressure overload-dependent membrane type I-matrix Cardiac-specific deletion of SOCS-3 prevents development of metalloproteinase induction: relationship to LV remodeling and left ventricular remodeling after acute myocardial infarction. Oba, fibrosis. Zile, M.R. et al. (2012) American Journal of Physiology, T. et al. (2012) Journal of the Heart and Circulation Physiology American College of Cardiology Used RT2 Profiler PCR Array for Human Extracellular Matrix and Adhesion Molecules Matrix metalloproteinases and LV remodeling Used a Custom RT2 Profiler PCR Array. Used RT2 Profiler PCR Array for Mouse Common Cytokines Title, Location, Date 17
  • Sample & Assay Technologies Application 1: Fibroblast collagen production and IL-17 Background and research question Fibroblasts from SSc patients show intrinsic TGF-beta1 activation, and other cytokines are also implicated in disease progression. Previous studies yielded conflicting reports on the association of IL-17 with SSc, and the authors sought to clarify its involvement. ? Are IL-17A&F and IL-17RA expressed differently in SSc vs. healthy subjects? Is IL-17 involved in regulating ECM during SSc? Nakashima, T. et al. (2012) J. Immunol. 188, 3573. 18
  • Sample & Assay Technologies Application 1: Fibroblast collagen production and IL-17 Approach Cytokines and receptors were measured in serum, fibroblast cultures, and tissue samples by ELISA, immunoblotting, and immunohistochemistry An RT2 Profiler PCR Array for Human Extracellular Matrix and Adhesion Molecules was used to profile ECM gene expression. An RT2 miRNA PCR Array was used to profile miRNA expression siRNA against TGF-beta1, Smad3, and IL-17RA were used to assess the effects of TGF-beta signaling on IL-17 receptor expression and IL-17 signaling on miR-129-5p expression, respectively. Nakashima, T. et al. (2012) J. Immunol. 188, 3573. 19
  • Sample & Assay Technologies Application 1: Fibroblast collagen production and IL-17 Major findings IL-17A levels were higher in sera and involved skin of SSc patients, and IL-17RA was lower at both the protein and mRNA level in cultured fibroblasts. This was rescued by siRNA for TGF-beta1 or Smad knockdown. The RT2 Profiler PCR Array showed that IL-17A treatment caused downregulation of pro-fibrotic CTGF. Alpha1(I) collagen expression remained the same, but was lower by immunoblotting. An RT2 miRNA PCR Array showed that miR-129-5p (among others) was downregulated in SSc fibroblasts. IL-17RA CTGF miR-129-5p collagen Nakashima, T. et al. (2012) J. Immunol. 188, 3573. Title, Location, Date 20
  • Sample & Assay Technologies Application 2: SOCS3 and myocardial infarction Fibrosis research with RT2 Profiler PCR Arrays Scleroderma and cytokines SOCS3 and myocardial infarction Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts. Nakashima, T. et al. (2012) Journal of Immunology Pressure overload-dependent membrane type I-matrix Cardiac-specific deletion of SOCS-3 prevents development of metalloproteinase induction: relationship to :LV remodeling left ventricular remodeling after acute myocardial infarction. Oba, and fibrosis. Zile, M.R. et al. (2012) American Journal of T. et al. (2012) Journal of the Physiology, Heart and Circulation American College of Cardiology Physiology Used RT2 Profiler PCR Array for Human Extracellular Matrix and Adhesion Molecules Used RT2 Profiler PCR Array for Mouse Common Cytokines Title, Location, Date Matrix metalloproteinases and LV remodeling Used a Custom RT2 Profiler PCR Array. 21
  • Sample & Assay Technologies Application 2: SOCS3 and myocardial infarction Rationale and research question Left ventricular remodeling after acute myocardial infarction (AMI), including fibrosis, contributes to heart failure. Previous work had shown that cytokines activating the JAK/STAT pathways could prevent LV remodeling in animal models after AMI. ? SOCS3 acts in a negative feedback loop induced by JAK/STAT-activating cytokines – could inhibition of SOCS3 prevent LV remodeling? Oba, T. et al. (2012) Am. Coll. Cardiol. 59, 838. 22
  • Sample & Assay Technologies Application 2: SOCS pathway Title, Location, Date 23
  • Sample & Assay Technologies Application 2: SOCS3 and myocardial infarction Approach Made cardiac-specific SOCS3 knockout mice, induced AMI, and observed LV remodeling in knockouts vs wild-types Performed western blot analysis, TUNEL staining for apoptosis, echocardiograph, and real-time PCR Used Mouse Common Cytokines RT2 Profiler PCR Array to profile cytokines in the system Oba, T. et al. (2012) Am. Coll. Cardiol. 59, 838. 24
  • Sample & Assay Technologies Application 2: SOCS3 and LV remodeling Major findings Survival was enhanced in SOCS3 knockouts after AMI induced by coronary ligation – 100% survived to 14 days, compared to 55% of controls LV remodeling was diminished in knockouts, as was apoptosis RT2 Profiler PCR Array showed expression of multiple JAK-STATactivating cytokines following AMI, and many were diminished in SOCS3 knockouts (including G-CSF, IL-11, and IL-6) Western blot showed greater activation of STAT3, AKT, and ERK pathways in knockouts Mallory-AZAN staining showed smaller fibrotic areas in knockout hearts, and MMPs, TGF-beta2, and collagen showed lower expression as well Conclusions Cardiomyocyte SOCS3 may drive fibrosis development/LV remodeling following AMI, and may be a useful therapeutic target. Oba, T. et al. (2012) Am. Coll. Cardiol. 59, 838. Title, Location, Date 25
  • Sample & Assay Technologies Application 3: MT1-MMP, LV remodeling, and fibrosis Fibrosis research with RT2 Profiler PCR Arrays Scleroderma and cytokines SOCS3 and myocardial infarction Matrix metalloproteinases and LV remodeling Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts. Nakashima, T. et al. (2012) Journal of Immunology Cardiac-specific deletion of SOCS-3 prevents development of left ventricular remodeling after acute myocardial infarction. Oba, T. et al. (2012) Journal of the American College of Cardiology Pressure overload-dependent membrane type I-matrix metalloproteinase induction: relationship to :LV remodeling and fibrosis. Zile, M.R. et al. (2012) American Journal of Physiology, Heart and Circulation Physiology Used RT2 Profiler PCR Array for Human Extracellular Matrix and Adhesion Molecules Used RT2 Profiler PCR Array for Mouse Common Cytokines Used a Custom RT2 Profiler PCR Array. Title, Location, Date 26
  • Sample & Assay Technologies Application 3: MT1-MMP, LV remodeling, and fibrosis Research question Myocardial fibrosis develops during chronic pressure-overload (PO), which causes LV hypertrophy Membrane type I MMP (MT1-MMP) is implicated in fibrosis development, and its transcription is enhanced by mechanical forces ? Could mechanical forces from chronic PO increase MT1-MMP expression and fibrosis? Zile, M. et al. (2012) Am. J. Physiol. Heart Circ. Physiol. 302, H1429 27
  • Sample & Assay Technologies Application 3: MT1-MMP, LV remodeling, and fibrosis Approach Developed an MT1-MMP promoter reporter mouse and used transverse aortic constriction to model PO Used a Custom RT2 Profiler PCR Array for MT1-MMP, procollagen type I, CTGF, TGF-betaR1, and other profibrotic genes in myocardial samples Measured LV by echocardiography and collagen by light microscopy Isolated papillary muscles from reporter mice and subjected to stimulation, then observed expression of MT1-MMP as well as transcription factors Zile, M. et al. (2012) Am. J. Physiol. Heart Circ. Physiol. 302, H1429 28
  • Sample & Assay Technologies Application 3: MT1-MMP, LV remodeling, and fibrosis Major findings PO led to LV hypertrophy and collagen volume fraction increase. MT1-MMP protein abundance increased over the course of 4 weeks after PO, and MT1-MMP promoter activity increased at 1 and 4 weeks, with a dip at week 2. The RT2 Profiler PCR Array showed increases in various profibrotic genes, including the TGF-beta receptor, collagens, serine protease inhibitors, LTBP, and CTGF, one week following PO. Increases in mechanical load led to strong increases in MT1-MMP expression in isolated papillary muscles, as well as expression of transcription factors including NFkappB, RELA, and c-Fos. Conclusions Mechanical forces during PO may activate MT1-MMP transcription via NFkappaB or c-Fos, exacerbating fibrosis through TGF-beta signaling. The temporal associations in this study suggest that further research into MT1MMP as a driver of LV remodeling is warranted. Title, Location, Date 29
  • Sample & Assay Technologies RT2 Profiler PCR Arrays for fibrosis research Interested in trying RT2 Profiler PCR Arrays? Visit us at www.sabiosciences.com/rt_pcr_product to learn more! Call 1-888-503-3187 for more information Email: support@SABiosciences.com (US & Canada) sabio@qiagen.com (International customer) Title, Location, Date 30